Cell viability was determined by MTT assay, the cells were treated with various concentrations of oxidative drug and mitochondrial toxins for 24 hours, then the MTT assay
6
was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dipheny-2H-tetrazolium bromide - MTT with DMEM were added per cultured cell, the optical density (OD) of culture well was measured by a microplate reader at 595 nm.
F
. ATP level measurement
ATP was monitored by detection of light caused by the reaction of ATP with added luciferase and D-luciferin. According to the manufacture’s instruction ATPlite™
Luminescence Assay System, 1000 Assay Kit PerkinElmer, cells were lysed with ATP Lit e lysis buffer (ATPLlite: NaOH, 100Mm Triton X100 4ml/L), with shaking and sonicatio n, centrifuge the cell lyste at 13,000rpm, 4oC, 5min, the supernatant was the samp h is a potentiometric, cell permeable fluorescent indicator that accumulates highly neg atively charged interior of mitochondria. The culture medium was removed from the adherent UB-OC1 cells, rinsed with PBS and resuspended in 500ul DMEM with 5ul of 1Mm TMRM(10uM final concentration), incubated at 37oC for 30mins. The cells were treated with CCCP for 10 min prior to TMRM staining. Fluorescence was quan
7
tified by flow cytometry (FACS Vantage, Becton Dickinson).
H. Reactive Oxygen species (ROS)
Intracellular ROS level was monitored by flow cytometry with 2’, 7’-dichlorodihyd rofluorescein (CM-H2DCFDA) fluorescent probe. The cells were rinsed with PBS prio r to pellet the cells, resuspended in 500ul PBS with 5uM CM-H2DCFDA, incubated at 37oC for 30mins. Fluorescence was quantified by flow cytometry (FACS Vantage, Becton Dickinson).
I. NAD
+/NADH ratio measurement
Whole cell NAD+ and NADH were measured using Elite Fluorimetric NAD/NADH ratio assay kit (Red Fluorescence) according to the manufacture’s instruction. Briefly Cells were lysed with NAD+ /NADH lysis buffer, with freeze thaw cycle and sonication, the lysate was centrifuged at 13,000rpm, 4oC for 15min, the supernatant was collected as the sample.
NAD+, NADH, NAD+/NADH ratio measurements were based on an enzymatic cycling
8 ormalized the with protein concentration.
K. Lactate production assay
9 ctrophoresis buffer. Then the gel was stained with developer solution: Lactate (3.24m g/ml), NAD+ (β-nicotinamide adenine dinucleotide 0.3mg/ml), NBT(Nitroblue Tetrazoliu m 0.8mg/ml), and PMS (Phenazine methosulfate 0.167mg/ml) dissolved in 10mM Tris -Hcl (pH8.5) buffer, incubated at 37oC for 30min, or longer time until saw the expec ted bands, stoppted the reaction by 5% acetic acid.
M. Western blot assay
Total proteins were extracted with the Rippa buffer. Protein concentration was assessed by Bio-Rad DC protein assay (Bio Rad Laboratories, Hercules.CA). Prot eins were separated by electrophoresis on 8%-12% sodium dodecyl sulfate polyacryla mide gels. Same amount of proteins (ug) were loaded in each lane. After electrophor esis, the proteins were transferred to the polyvinylidene difluoride membrane (PVDF) or nitrocellulose membrane (NC) and subsequently subjected to immunoblottin g analysis using appropriate antibodies. The amount of loading was further detected b y western blotting housekeeping protein (Lamin A, 1:5000 dilution). Using horseadish
10
peroxidase - conjugated secondary antibody. Protein bands on the blots were visualized by ECL western blot detection reagent.
All results are the average of at least three independent samples.
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Ⅲ . RESULTS
A. Generation of whole body LDHB knock out mouse
Our lab generated whole body LDHB gene knock out mouse as inactivation of LDHB gene by Cre/Loxp system. The conditional mouse harbors the floxed allele, in which LDHB gene has flanked by two Loxp sites. The LDHB gene can be exc ised and inactivated by Cre recombinase recognizing two Loxp sites.
Two mouse lines were required for conditional gene-LDHB deletion. First, a c onventional transgenic mouse line with Cre targeted to protamine in testis. Secondl y a mouse strain that embodies a target gene–LDHB flanked by two Loxp sites. R ecombination occurs only in those cells expressing Cre recombinase. Thus, the LD HB remains active in all cells and tissues which do not express Cre. We successfu lly generated whole body LDHB knock out mouse, which express the loxP and Cr e. LDHB expression protein level and LDH isozyme in heart tissue (Figure1, A). Th e mouse contains only loxP but not cre was the wild type mouse which we genera ted for control group.
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Figure1. Generation of the LDHB knockout mouse. Restriction map of the 5’
region of the wild-type LDHB locus (wild-type locus), the targeting vector, the LDHB locus after homologous recombination (Targeted/neo locus), the LDHB locus after Flp-recombinase-mediated excision of PGK-neo gene, and knockout LDHB locus (knock out locus) (A), analysis of control (C) and targeted (T) ES cells. Digestion of ES cell DNA with BamH1 generates a novel fragment of 7.5 kb in (Targeted/neo locus) clones (B), protein level of LDHB expression in LDHB knock out mouse heart tissue(C), LDH isozyme pattern in LDHB knock out mouse heart tissue (D).
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B. LDHB knock out mouse grows normally
LDHB is important in maintaining high level of pyruvate and highly expressed in hig h energy demanding tissues such as heart and kidney. In response to knock out, the LDH activity from lactate side of reaction (lactate to pyruvate: L-P) showed clear decrease due to deficiency of LDHB in total activity. The LDH activity from pyruvate side of reaction (pyruvate to lactate: P-L) showed decrease of total LDH activity as well, it mainly because in LDHB KO mouse the LDHA level in protein level was not changed, therefore the total LDH was decreased by LDHB deficiency(Fig.2A). To confirm whether loss of LDHB gene aff ects on the growth of mouse, we measured the body weight of LDHB knockout mous e in male and female groups for 48weeks. The LDHB knock out mice in male and female both groups showed normal growth (Fig2B).
14 Fig.2. LDHB knock out mouse grows normally.
LDH activities were decreased in heart and kidney of LDHB knockout mouse (A), L DHB knockout mouse grows normally (B). White bars and circles represent control mice, and black bars and circles represent Knockout mice. *p<0.05,**p<0.01,***p<0.0 01 vs. wild type by Student’s t-test.
15 use, we conducted the brainstem response (ABR), a common electrophysiological test of hearing function. The results showed the age-dependent increase of hearing threshol ds were observed at high (32kHz) frequency region and middle (16kHz) frequency region i n 10 month old wild type mice (Fig.3C). Interestingly, in LDHB knock out group incr
16
A B C
Fig.3.LDHB knock out mouse show early onset of age related hearing loss.
Auditory brainstem response (ABR) hearing threshold test with 2 months(A), 5 month s and 10 months old Wild type and LDHB Knockout mice at 8kHz, 16 kHz and 32 kHz three different frequencies. The wild type mice has hearing threshold increase at the age of 10 months(C). Increase of auditory threshold was observed in knock out mice at the age of 5 month(B), at the age of 10 months, both wild type and knock out mice showed hearing loss, the knock out mice showed more severe hearing threshold increase than wild type. *p<0.05,**p<0.01,***p<0.001 vs. wild type by Stud ent’s t-test.
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D. Differentiated hair cell express LDHB and show increased m itochondrial function
Hair cell damage with ROS production related mitochondrial dysfunction is thought to be the essential role of age-related hearing loss (Hengchao, Chen et al; 2014). In vivo study we observed the LDHB knockout mouse has the high frequency hearing threshold increase and hair cell loss earlier than wild type group. To investigates the molecular mechanism of hair cell loss in LDHB knock out mouse. UB-OC1 cell as hair cell precursor was app lied for further study. It differentiates to the hair cell at 37oC, 5%CO2 (Hasson et al., 1997). UB-OC1 cell up-regulated the cyto-skeletal protein Myosin VIIa as hair cell m arker and down regulated the supporting cell marker Jagged1 under differentiating co ndition(Fig,4A). We determined the expression level of LDHA and LDHB (Fig, 4A).
The clear increase of LDHB was observed in differentiated UB-OC1 which mainly contain LDH1 and LDH2, but there was no change in LDHA expression compare wi th proliferative UB-OC1 (Control group). To investigate the affect of LDHB induction on mitochondrial function we investigated two critical indicators of energy status: the i ntracellular ATP and NAD+/NADH levels, differentiated UB-OC1 had higher ATP lev el (Fig, 4C). Moreover, increased expression of mitochondrial respiratory proteins (Fig, 4D), mitochondria in RNA level increase (Fig, 4F), companied with mitochondrial me mbrane potential increase (Fig, 4F) were observed as well. Indicates the mitochondrial function was increased after differentiation. With increase of mitochondrial respiratory chain activity the more electron donor NADH convert to NAD+ therefore the redox state NAD+/N ADH level (Fig, 4G) increased in differentiated UB-OC1.
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It is widely known that mitochondria, beside play the critical role in aerobic ATP generation and in different pathways, are the primary source of ROS as well. We found that the intracellular ROS production had increased (Fig, 4H) in differentiated UB-OC1.
To examine whether the mitochondrial function increase was related to LDH isozyme changes, the LDH isozymes of the UB-OC1 cells were analyzed using a native PAGE gel electrophoresis assay. Interestingly, the differentiated UB-OC1 showed a distinct LDH isozyme pattern (Fig, 4B), they retained LDH1 and LDH2, which normally highly ex pressed in energy demanding tissue, whereas proliferative UB-OC1 retains mostly LD H5. Differentiated UB-OC1 had higher LDH activity (Fig, 4I) and, correlatively, relea sed greater amount of lactate production (Fig, 4J) into the media than proliferative UB -OC1. These results suggest that differentiated UB-OC1 express LDHB and there is c lear increase of mitochondrial function.
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Fig.4. Increased LDHB expression and mitochondrial function observed in differe ntiated UB-OC1 cell.
Hair cell precursor UB-OC1 cell undergoes differentiation at 37oC, 5% CO2. LDHB e xpression (A), ATP level (C), mitochondrial respiratory subunits (D), Mitochondrial R NA levels (E), Mitochondria membrane potential (F) NAD+/NADH levels (G),ROS le vel(H), LDH activity(I), Lactate production in media (J) are increased in differentiated UB-OC1 cells. The isozymes pattern shift to LDH1, LDH2 in differentiated UB-OC1.
White bars represent control cells and black bars represent differentiated cells. *p<0.0
20
5,**p<0.01,***p<0.001 vs. control( proliferative UB-OC1) by Student’s t-test.
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E. LDHB knockdown decreased the mitochondrial function in differentiated UB-OC1
To investigate the mechanism of hair cell loss in LDHB deficient condition, we studied mitochondrial function in LDHB knockdown differentiated UB-OC1. In LDHB knockdown UB-OC1 cell, LDHB expression was decreased in the protein level (Fig.5A), without changes in LDHA expression (Fig.5A). It companied with LDH isozymes pattern shift (Fig.5B) toward contains LDH5 mainly. We observed deficiency of LDHB cause the ATP production decrease (Fig.5D), mitochondrial membrane potential (Fig.5F) N AD+/ N ADH level decrease (F ig. 5G). Even though there was no clear change in mitochondrial respiratory subunits. We concluded deficiency of LDHB causes the mitochon drial dysfunction in differentiated UB-OC1 cell. It companied by the intracellular ROS pr oduction increase (Fig.5H). The LDHB knock down cell showed lower LDH activity (Fig.
5C), the result is consistent with LDH activity in the heart and kidney tissue. But there was no clear change was observed in the release amount of lactate production into the m edia compare with control siRNA group (Fig.5I).Collectively, these results explain that LDHB knock down decreased mitochondrial function in differentiated UB-OC1 cell.
22
Fig.5. Decreased mitochondrial function in LDHB knockdown differentiated UB-O C1 cell.
LDHA and LDHB expression (A), LDH activity (C), ATP level (D), mitochondrial m embrane potential (F), NAD+/NADH ratio (G) are decreased in differentiated UB-OC1 cells. There is no change in LDHA expression (A) and Mitochondrial respiratory sub units (E). ROS level (H) is increased in differentiated UB-OC1. Lactate level in medi a is no significant change in differentiated UB-OC1 (I). White bars represent control cells and black bars represent LDHB knockdown cells. *p<0.05,**p<0.01,***p<0.001 vs. control siRNA by Student’s t-test.
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F. L D H B k n o c k d o w n differentiated UB-OC1 c e l l s stabilized the Hif1a expression.
Recent study shows that decreased levels of NAD+ contribute to the mitochondrial dysfunction related with Hif1a stabilization, skeletal muscle aging and that sirtuin 1 (SIRT1)modulates (Gomes et al., 2013) . In our study deficiency of LDHB in differentiated UB-OC1 showed the decreased level of NAD+/NADH and reduced mitochondrial function. To investigate the mechanism of LDHB deficiency induced mitochondrial dysfunction, we checked the Hif1a expression in LDHB knockdown differentiated UB-OC1. The Hif1a expression was stabilized in response of LDHB knockdown in differentiated UB-OC1 (Fig6). Thus LDHB knock down decrease NAD
+ and stabilize HIF1a which probably cause mitochondrial dysfunction in differentiated UB-OC1.
Fig.6. Induced Hif1a expression in LDHB knockdown differentiated UB-OC1 cells.
Hif1a expression in LDHB knock down differentiated UB-OC1.
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G. LDHB knockdown differentiated UB-OC1 cells are sensitive to mitochondrial toxins
We observed clear mitochondrial defects in the response of LDHB deficiency in hair cell. The mtDNA mutations/deletions, ROS production related mitochondrial dysfunctions are thought to be an essential component of the age-associated disorder in auditory sensitivity (T. Yamasoba et al., 2007). To investigate the mitochondrial toxin affect on LDHB deficient hair cell. The test on respiratory inhibitors, such as Rotenone (a complex I inhibitor), CCCP (mitochondrial uncoupling agent), Antimycin a (a complex III inhibitor) and Oligomycin (a complex V inhibitor) were studied. We measured the cell viability by MTT assay with LDHB knockdown UB-OC1 cells which treated with the respective doses of inhibitors for 24 hours. LDHB knockdown UB-OC1 group showed less cell viability compare with control group (Fig.7A).Cisplatin is a widely used chemotherapeutic agent to treat variety of cancer. But it shows the high does regimens ototoxicity as side affect. It mainly damages the outer hair cells in the basal turn, spiral ganglion neuron cells and the stria vascularis, resulting in hearing loss. The mechanisms appear to involve the production of reactive oxygen species (ROS), which can trigger cell death (Rybak L et al., 2006). Cisplatin accumulates in mitochondria causes a direct and significant impairment of mtDNA and mtRNA thesis (Nuria GARRIDO et al., 2008). Gentamicin is one of most common applied antibiotic treat variety of bacterial infections, particularly those caused by Gram-negative organisms.
25
Gentamicin toxic to the sensory cell in ear, gentamicin -induced ototoxicity is usually bilateral, symmetrical, irreversible and typically affects the higher frequencies (J.
Schacht 1998). LDHB deficiency increased sensitivity to cisplatin, gentamicin treatment in hair cell. These results explained the LDHB knockdown induces the sensitivity to the mitochondrial toxin and antibiotics, which is important clinical information for future study.
26
Fig.7.LDHB knockdown increases sensitivity to mitochondrial toxins in differe ntiated UB-OC1 cell.
100uM of H202 ; 2.5uM of Cisplatin; 1mM Gentamicin; 5uM of Rotenone; 10uM of CCCP; 5uM of Antimycin a; 1uM of Oligomycin a were respectively treated on differentiated UB-OC1 for 24 hours, there are decrease of cell viability in LD HB knock down UB-OC1 group. White bars represent control cells and black bar s represent LDHB knockdown cells. *p<0.05,**p<0.01,***p<0.001 vs. control siR NA by Student’s t-test.
27
Ⅳ . Discussion
Mitochondrial pathology plays important role in different type of hearing loss (Pickles. J.O et al., 2004). Acquired mitochondrial dysfunction in hearing loss seems to involve increased ROS and decreased energy production, redox imbalance. It is well known that pyruvate as the substrate of the mitochondrial ATP production, is the fina l product of glycolysis. LDHB is important in maintaining high level of pyruvate lev el, LDHB expression could be directly correlated to mitochondrial function and energ y production.
A widely accepted hypothesis of how aging leads to age-related hearing loss is through mitochondrial dysfunction related with reactive oxygen species (ROS), mtDNA mutants and ATP deficiency plays a key role in aging process of cochlear cells (Shinic hi Someya et al,.2010)..
In this study, first we show that the deficiency of LDHB gene causes the early onset of age related hearing loss, even though the growth is normal in mouse. LDHB KO mouse lost hearing ability earlier than WT, even though it lost hearing ability in high frequency sound, while low frequency is preserved, which companied hair cell loss, spiral ganglion neuron cell and lateral wall degradation in high frequency region. Hence, these results suggest that LDHB play a critical role in cochlear aging, AHL in general. In response of knocking out LDHB, the total LDH activity in both side reaction: lactate side of reaction (lactate to pyruvate: L-P) and pyruvate side of reaction (pyruvate to lactate: P-L) were decreased (Fig.2A). Based on previous study (Krieg AF et al., 1967) and our experimental
28
observation the LDHB is highly sensitive to substrate inhibition. In higher concentration of pyruvate, LDHB activity is inhibited by pyruvate. In our experiment we selected the 0.5mM of pyruvate as substrate which avoided the substrate inhibition. In addition LDHB KO mouse the LDHA level in protein level was not changed, therefore the total LDH activity was decreased by LDHB deficiency in pyruvate side of reaction as well, which constant with cell line study result (Fig.5C).
We found that enhance of LDHB mediates the increase of mitochondrial function followed an enhanced NAD+/NADH balance, ATP level, mitochondrial respiratory chain subunits (Fig4.G,C,D).Mitochondrial respiratory chain catalyzes the NADH oxidation. The increase of mitochondrial respiratory chain activity elevates the NAD+/NADH ratio and translocates protons across the inner mitochondrial membrane, which ultimately leads to increase of ATP production.
The deficiency of the LDHB causes the mitochondrial dysfunction in hair cell of cochlear, triggered decline in NAD+ /NADH level (Fig5.G), stabilization of the Hif1a expression (Fig.6). The decreased NAD+ and the accumulation of HIF-1α under normoxic conditions which probably cause mitochondrial dysfunction in LDHB knock down mouse.
Tissues that are composed of postmitotic cells such as the brain and the inner ear are particularly vulnerable to mitochondrial dysfunction because of their high energy requirements, and inability to undergo regeneration.
We observed mitochondrial dysfunction in the response of LDHB deficiency in hair cell.
Mitochondrial dysfunctions are thought to be an essential component of the age-associated disorder in auditory sensitivity (Fig.7). With LDHB deficiency, the hair cell becomes more
29
sensitive to the mitochondrial toxin. We postulated that the deletion of LDHB leading the mitochondrial dysfunction and related increase of oxidative stress accelerates the hair cell damage with mitochondrial toxin challenge.
Cisplatin and Gentamicin are most commonly applied drug in clinic, unfortunately both of them show ototoxicity as side affect (Leonard Rybak et al., 2006), induce the oxidative stress and mitochondrial defects in cochlear of inner ear. In our results suggested that LDHB deficiency results the mitochondrial defects which increases the sensitivity to cisplatin, gentamicin treatment in hair cell (Fig.7). These postulated the LDHB knockdown induces the sensitivity to the mitochondrial toxin and antibiotics, which is important clinical information for future study.
30
V. Conclusion
In summary, the deficiency of LDHB gene causes the early onset of age related hearing loss. LDHB knock down decreased NAD+ level and stabilized Hif1a which pro bably cause mitochondrial dysfunction in hair cell. LDHB knock down sensitized hair cell to antibiotics which support important clinical information.
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